Needlestick injury is relatively common amongst healthcare workers, particularly those, such as anaesthetists, who regularly perform invasive procedures. The risk of seroconversion following needlestick injury may be reduced by knowledge of body fluids that are high risk and knowledge of post-exposure prophylaxis following possible HIV-contaminated needlestick injury. A structured questionnaire was used to establish knowledge regarding high HIV risk body fluids and measures to be taken following needlestick injury in anaesthetists working in a large teaching hospital. Completed questionnaires were obtained from all 76 anaesthetists working in the department (39 consultant, 37 trainee/non-consultant). Only 45.2% correctly identified high-risk body fluids. Sixty-eight per cent of anaesthetists knew the appropriate first aid measures to be taken following needlestick injury. Only 15% of anaesthetists were aware that post-exposure prophylaxis (oral medication) should be administered within 1 h of injury. This study reveals a surprisingly poor knowledge of high-risk body fluids and action to be taken following needlestick injury. Timely post-exposure prophylaxis, after needlestick exposure to high-risk body fluids, is believed to reduce the risk of seroconversion to HIV. Ignorance of this may increase the risk of seroconversion to HIV for anaesthetists and other healthcare professionals.